Methods for regulating cancer

ABSTRACT

The present invention provides methods for inhibiting cancerous growth of a cell using an antibody or an antigen-binding portion thereof which binds to an epitope of EphB4 polypeptide. Purified antibodies of EphB4 are also provided. The invention also provides methods for preventing or treating cancer. The invention also relates to methods of identifying agents that can inhibit cancerous growth of a cell.

FIELD OF THE INVENTION

The present invention relates to methods for regulating cancer. Inparticular, the invention relates to methods for inhibiting cancerousgrowth of a cell. The invention also provides methods for preventing ortreating cancer. The invention also relates to methods of identifyingagents that can inhibit cancerous growth of a cell.

BACKGROUND OF THE INVENTION

Cancer describes a range of diseases, which result from dysregulatedgrowth of cells of the body. Malignant cancers may develop from thisdysregulated growth and subsequently spread around the body via thebloodstream or the lymphatic system, a process known as metastasis.Malignant tumours of epithelial tissues are the most common form ofcancer and are responsible for the majority of cancer-related deaths inwestern industrialised countries. According to the Australian Instituteof Health and Welfare (AIHW), on average one in three men and one infour women will develop cancer before the age of 75 years (1). In menthe most common cancers are prostate, bowel and lung and in women,breast, bowel and melanoma. Identification of genes expressedspecifically in tumour tissues and not in normal tissues, and analysisof their functions are useful for identifying new targets for cancertherapy.

Several genes have been implicated in various cancers. For instance,oncogenes are known to code for receptors for cellular growth factorsuch as epidermal growth factor. The ras gene is an oncogene that isbelieved to be responsible for up to 90% of all human pancreatic cancer,50% of human colon cancers, 40% of lung cancers, and 30% of leukemias.Mutated oncogenes can become cancer-causing genes. Such mutatedoncogenes code for proteins such as protein kinases and proteinphosphorylating enzymes that trigger uncontrolled cell growth. EphB4 isa recently identified member of the largest known family of receptorprotein tyrosine kinases. Eph receptor family members have beenidentified to be involved in many cellular processes including neuraldevelopment, angiogenesis and vascular network assembly (2-5). As aresult of interactions with their ligands, the ephrins, they mediatecontact-dependent cell interactions, which regulate cell functions suchas contact inhibition, cytoskeletal organisation and cell migration (6,7).

Although a number of anti-cancer agents including growth inhibitorymolecules such as cytoxic compounds have been developed in an attempt totreat cancer, there still remains a need for providing effective methodsfor regulating cancer.

SUMMARY OF THE INVENTION

The present invention is based on the surprising finding that anantibody that can bind to a particular region of the EphB4 protein canadvantageously inhibit cancerous growth in a cancer cell by causing celldeath of the cancer cell.

Therefore, in a first aspect the present invention provides a method forinhibiting cancerous growth of a cell, the method comprising contactingthe cell with at least one antibody or an antigen-binding portionthereof, wherein the antibody or antigen-binding portion thereof bindsto an epitope located within residues 200 to 400 of EphB4 (SEQ ID NO:1), or a sequence at least 85%, preferably at least 90% identicalthereto. Preferably, the antibody or antigen-binding portion thereofbinds to an epitope located within residues 201 to 245 of EphB4 (SEQ IDNO: 1), or a sequence at least 85%, preferably at least 90% identicalthereto. Preferably, the antibody or antigen-binding portion thereofbinds to an epitope located within residues 220 to 244 of EphB4 (SEQ IDNO: 1), or a sequence at least 85%, preferably at least 90% identicalthereto. Most preferably, the antibody or antigen-binding portionthereof binds to an epitope located within residues 220 to 230 of EphB4(SEQ ID NO: 1), or a sequence at least 85%, preferably at least 90%identical thereto. Preferably, the sequence has a substitution of aminoacid Asp (D) to Asn (N) at residue 226 of EphB4 (SEQ ID NO: 1).

In a second aspect the present invention also provides a method forinducing cell death of a cancer cell, the method comprising contactingthe cell with at least one antibody or an antigen-binding portionthereof, wherein the antibody or antigen-binding portion thereof bindsto an epitope located within residues 200 to 400 of EphB4 (SEQ ID NO:1), or a sequence at least 85%, preferably at least 90% identicalthereto. Preferably, the antibody or antigen-binding portion thereofbinds to an epitope located within residues 201 to 245 of EphB4 (SEQ IDNO: 1), or a sequence at least 85%, preferably at least 90% identicalthereto. Preferably, the antibody or antigen-binding portion thereofbinds to an epitope located within residues 220 to 244 of EphB4 (SEQ IDNO: 1), or a sequence at least 85%, preferably at least 90% identicalthereto. Most preferably, the antibody or antigen-binding portionthereof binds to an epitope located within residues 220 to 230 of EphB4(SEQ ID NO: 1), or a sequence at least 85%, preferably at least 90%identical thereto. Preferably, the sequence has a substitution of aminoacid Asp (D) to Asn (N) at residue 226 of EphB4 (SEQ ID NO: 1).

In a third aspect the present invention provides a method for treatingor preventing cancer in a subject, the method comprising administeringto the subject an effective amount of at least one antibody or anantigen-binding portion thereof, wherein the antibody or antigen-bindingportion thereof binds to an epitope located within residues 200 to 400of EphB4 (SEQ ID NO: 1), or a sequence at least 85%, preferably at least90% identical thereto. Preferably, the antibody or antigen-bindingportion thereof binds to an epitope located within residues 201 to 245of EphB4 (SEQ ID NO: 1), or a sequence at least 85%, preferably at least90% identical thereto. Preferably, the antibody or antigen-bindingportion thereof binds to an epitope located within residues 220 to 244of EphB4 (SEQ ID NO: 1), or a sequence at least 85%, preferably at least90% identical thereto. Most preferably, the antibody or antigen-bindingportion thereof binds to an epitope located within residues 220 to 230of EphB4 (SEQ ID NO: 1), or a sequence at least 85%, preferably at least90% identical thereto. Preferably, the sequence has a substitution ofamino acid Asp (D) to Asn (N) at residue 226 of EphB4 (SEQ ID NO: 1).

In another aspect of the invention there is provided a method ofidentifying an agent which inhibits cancerous growth of a cell, themethod comprises assessing the ability of the agent to bind to an EphB4polypeptide within the region of residues 200 to 400 of EphB4 (SEQ IDNO:1), or a sequence at least 85%, preferably at least 90% identicalthereto. Preferably, the method comprises assessing the ability of theagent to bind to an EphB4 polypeptide within the region of residues 201to 245 of EphB4 (SEQ ID NO: 1), or a sequence at least 85%, preferablyat least 90% identical thereto. Preferably, the method comprisesassessing the ability of the agent to bind to an EphB4 polypeptidewithin the region of residues 220 to 244 of EphB4 (SEQ ID) NO: 1), or asequence at least 85%, preferably at least 90% identical thereto. Mostpreferably, the method comprises assessing the ability of the agent tobind to an EphB4 polypeptide within the region of residues 220 to 230 ofEphB4 (SEQ ID NO: 1), or a sequence at least 85%, preferably at least90% identical thereto. Preferably, the sequence has a substitution ofamino acid Asp (D) to Asn (N) at residue 226 of EphB4 (SEQ ID NO: 1).

The present invention also provides an agent identified by the methoddescribed above.

In a further aspect of the invention there is provided a purified EphB4antibody which binds to a polypeptide having a sequence at least 85%identical to residues 201 to 245 of EphB4 (SEQ ID NO: 1), preferably atleast 90% identical to residues 201 to 245 of EphB4 (SEQ ID NO: 1).Preferably, the purified EphB4 antibody binds to a polypeptide having asequence at least 85% identical to residues 220 to 244 of EphB4 (SEQ IDNO: 1), preferably at least 90% identical to residues 220 to 244 ofEphB4 (SEQ ID NO: 1). The purified EphB4 antibody preferably binds to apolypeptide having a sequence at least 85% identical to residues 220 to230 of EphB4 (SEQ ID NO: 1), preferably at least 90% identical toresidues 220 to 230 of EphB4 (SEQ ID NO: 1). Most preferably, thepresent invention provides a purified EphB4 antibody which binds to anepitope located in residues 200 to 400 of EphB4 (SEQ ID NO: 1). Thepurified EphB4 antibody according to the present invention preferablybinds to a polypeptide having a substitution of amino acid Asp (D) toAsn (N) at residue 226 of EphB4 (SEQ ID NO: 1). Preferably, the purifiedEphB4 antibody is a monoclonal antibody.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows immunohistochemical localisation of EphB4 expression inthree different colon cancers and matched normal mucosa using the EphB4polyclonal antibody (H-200-Santa Cruz Biotechnology) specificallydirected to the extracellular domain amino residues 201 to 400 of EphB4(SEQ ID NO:1). The dark stain from the biotinylated secondary antibodyindicates the EphB4 protein. Nuclei are stained with Harrishaematoxylin. High power (100×) magnification images of three differentadenocarcinomas (well differentiated, moderately well differentiated andpoorly differentiated) and their matched normal mucosa are shown. Strongstaining of the tumour tissue and very weak, diffuse staining of normaltissue was evident for each sample set. There was no cross-reactivitywith the secondary antibody alone (result not shown).

FIG. 2 shows relative RT-PCR comparing expression of EphB4(1187 bp) andinternal 18S rRNA (489 bp) in five tumour (T)/normal (N) pairs. LM—Livermetastasis and NL—normal liver from patients 5, C1—colon cancer cellline LIM2405, C2—colon cancer cell line SW480, RT—RT negative control,P—PCR negative control, M—pUC19/HpaII marker.

FIG. 3 shows a confluent monolayer of cells in 2 ml DMEM is treated witha 1/500 dilution of the EphB4 polyclonal antibody (H-200-Santa CruzBiotechnology) specifically directed to the extracellular domain aminoresidues 201 to 400 of EphB4 (SEQ ID NO:1) (0.4 ng/μl finalconcentration). Incubation of cells with the antibody causes a visualresponse between 24 and 48 h. The cells lift off the bottom of theculture vessel in a fragile sheet which breaks up easily on gentleagitation. The change in colour of the media is due to a 0.5 pH unitchange attributed to the leakage of cellular contents from dead cells.

FIG. 4 shows a graph showing effect of increased doses of an EphB4polyclonal antibody (H-200-Santa Cruz Biotechnology) specificallydirected to the extracellular domain amino residues 201 to 400 of EphB4(SEQ ID NO:1) on growth of MCF-7 cells in vitro after 48 hours.

FIG. 5 shows a trypan blue exclusion assay to determine dose dependencyof an EphB4 polyclonal antibody (H-200-Santa Cruz Biotechnology)specifically directed to the extracellular domain amino residues 201 to400 of EphB4 (SEQ ID NO:1). The number of viable cells (x100000)(Y-axis) decreases after 48 h exposure to three different dilutions ofthe EphB4 antibody (X-axis). There were no viable cells remaining after72 h with the addition of 1 μg/ml and after 48 h with the addition of 2μg/ml.

FIG. 6 shows a Caspase-3 assay. The relative amount of caspase-3activity per 100000 cells (Y-axis) increases after time for alldilutions of an EphB4 polyclonal antibody (H-200-Santa CruzBiotechnology) specifically directed to the extracellular domain aminoresidues 201 to 400 of EphB4 (SEQ ID NO:1) (X-axis). Because there wereno viable cells counted for 1/200 dilution (1 μg/ml) after 72 h and forthe 1/100 (2 μg/ml) dilution after 48 h, caspase-3 activity was notmeasured.

FIG. 7 shows a graph showing percentage viability of breast cancer cellsafter 65 h treatment with five different EphB4 antibodies: (1) a EphB4polyclonal antibody (Swiss) directed to amino acid residues 825 to 991of the carboxy terminus of mouse EphB4 (gift from Dr Andrew Ziemiecki,University of Bern), (2) a polyclonal N-terminal EphB4 antibody (N-19Santa Cruz Biotechnology) directed to the N-terminal first 19 aminoacids of the EphB4 amino acid sequence which is likely to be amino acidsresidues 16 to 34 of the precursor EphB4 (SEQ ID NO:1), (3) a polyclonalEphB4 C-terminal antibody (C-16 Santa Cruz Biotechnology) directed tothe carboxy-terminal corresponding to tyrosine kinase domain consistingof amino acid residues 615 to 874 of EphB4 (SEQ ID NO:1), (4) a EphB4polyclonal antibody (H-200-Santa Cruz Biotechnology) specificallydirected to the extracellular domain amino residues 201 to 400 of EphB4(SEQ ID NO:1) and (5) EphB4 polyclonal antibody (H-200 (old)-Santa CruzBiotechnology-Lot number B141 batch) specifically directed to theextracellular domain amino residues 201 to 400 of EphB4 (SEQ ID NO:1).Cells were treated with 1/100 dilution of stock antibody (200 μg/ml),then stained with trypan blue (stains dead cells). Ratios of unstained(viable) to stained (unviable) were calculated for four differentaliquots of each treatment. Control—no antibody added. CLM—complementlimited medium. FCS—10% Fetal calf serum added to medium. Complementdoes not play a role in the cell death effect of the EphB4 polyclonalantibody (H-200-Santa Cruz Biotechnology) specifically directed to theextracellular domain amino residues 201 to 400 of EphB4 (SEQ ID NO:1).This is demonstrated by the comparison of percentage viability afterantibody addition to cells grown in medium with normal protein activity(FCS experiment) with cells grown in medium in which complement proteinswere inactivated by heating to 55° C. for 30 mins (CLM Experiment).

FIG. 8 shows a western analysis of normal human tissues as indicated anda representative colon tumour (Tumour). (A) EphB4 protein was identifiedusing a EphB4 polyclonal antibody (H-200-Santa Cruz Biotechnology)specifically directed to the extracellular domain amino residues 201 to400 of EphB4 (SEQ ID NO:1). Predicted wildtype protein is 120 kDa and isindicated by the arrow. (B) Coomassie blue stained duplicate gel. Sizesof molecular weight marker are as indicated.

FIG. 9 shows a schematic diagram comparing the domains of the wild-typeEphB4 receptor with the predicted structure of the slice variantsEphB4v1 and EphB4v2. Deleted regions are indicated by *.

FIG. 10 shows an expression of EphB4gene and PBGD (housekeeping control)in breast cancer cell lines MDA-MB-231 (231), MDA-MB-468 (468), Hs578t,MCF7 and T47D either treated with 1/100 dilution of the EphB4 polyclonalantibody (H-200-Santa Cruz Biotechnology) specifically directed to theextracellular domain amino residues 201 to 400 of EphB4 (SEQ ID NO:1)for 65 h (Treated) or with no treatment (control). RT-ve is the reversetranscription reagents only control. Marker 1-Spp1/EcoR1 and Marker2-pUC19/Hpa11.

FIG. 11 shows the sequences of the six overlapping peptides [shown asSEQ ID NO:2 (Peptide 1) to SEQ ID NO:7 (Peptide 6)] designed to span thefirst 125 amino acids of the target EphB4 sequence (shown in bold). Thenumbers refer to the position of the amino acids in the precursor EphB4protein (SEQ ID NO:1 shown in FIG. 18).

FIG. 12 shows a trypan blue exclusion assay comparing viability incontrol cells (untreated), cells with the EphB4 polyclonal antibody(H-200-Santa Cruz Biotechnology) specifically directed to theextracellular domain amino residues 201 to 400 of EphB4 (SEQ ID NO:1)alone (Ab only) or treated with both antibody and peptide cocktail(Ab+all peptides). Tests were performed in duplicate.

FIG. 13 shows an assay comparing relative levels of caspase-3 activityin control cells (untreated), cells with the EphB4 polyclonal antibody(H-200-Santa Cruz Biotechnology) specifically directed to theextracellular domain amino residues 201 to 400 of EphB4 (SEQ ID NO:1)alone, (Ab only) or treated with both antibody and peptide cocktail(Ab+all peptides). Tests were performed in duplicate.

FIG. 14 shows results of trypan blue exclusion assay performed 48 hafter treatment on confluent SW480 monolayers with a 1/500 dilution ofthe EphB4 polyclonal antibody (H-200-Santa Cruz Biotechnology)specifically directed to the extracellular domain amino residues 201 to400 of EphB4 (SEQ ID NO:1) with or without 5 μl of peptides asindicated. Peptides 1 and 2 appeared to rescue ˜50% of cells fromAb-mediated cell death.

FIG. 15 shows an increase in the volume of Peptide 1 (SEQ ID NO:2) orPeptide 2 (SEQ ID NO:3) (to 10 μl) was able to fully rescue cells fromthe EphB4 polyclonal antibody (H-200-Santa Cruz Biotechnology)specifically directed to the extracellular domain amino residues 201 to400 of EphB4 (SEQ ID NO:1) mediated cell death and was equal in effectto a combination of Peptides 1 and 2 (5 μl of each).

FIG. 16 shows the sequences of the two overlapping peptides [shown asSEQ ID NO:2 (Peptide 1) and SEQ ID NO:3 (Peptide 2)] that were able toblock the function of the EphB4 polyclonal antibody (H-200-Santa CruzBiotechnology) specifically directed to the extracellular domain aminoresidues 201 to 400 of EphB4 (SEQ ID NO:1) on cells in culture andsequences of three peptides [shown as SEQ ID NO:8 (Peptide 7) to SEQ IDNO:10 (Peptide 9)] designed about the core sequence GSCVV for furthernarrowing of the reactive sequence. The numbers refer to the position ofthe amino acids in the precursor EphB4 protein (sequence shown in boldfont).

FIG. 17 shows Peptide 7 (SEQ ID NO: 8) was able to fully rescue cellsfrom Ab-mediated cell death and was equal in effect to Peptide 2 (SEQ IDNO: 3). One microlitre of the EphB4 polyclonal antibody (H-200-SantaCruz Biotechnology) specifically directed to the extracellular domainamino residues 201 to 400 of EphB4 (SEQ ID NO:1) (0.2 μg/ml) and 10 μlof a 10 mg/ml stock of peptide were pre-incubated together on ice for 2h before addition to a confluent monolayer of cells in a 24-wellmicrotitre plate with 500 μl of DMEM.

FIG. 18 shows the amino acid sequence of SEQ ID NO:1. SEQ ID NO:1 is theamino acid sequence of precursor Homo sapiens Ephrin type-B receptor 4(EphB4).

FIG. 19 shows a model of the EphB4 receptor with domain regions to whichthe different polyclonal antibodies (N-19, H-200 and C-16) have beentargeted indicated by black brackets. The globular domain (ephrinreceptor ligand binding domain) corresponds to residues 29 to 197 ofEphB4 (SEQ ID NO:1). The cysteine-rich region (Giardia variant-specificsurface protein) corresponds to residues 255 to 313 of EphB4 (SEQ IDNO:1). The fibronectin type III domain 1 corresponds to residues 324 to414 of EphB4 (SEQ ID NO:1). The fibronectin type III domain correspondsto residues 437 to 517 of EphB4 (SEQ ID NO:1). The transmembrane domaincorresponds to residues 540 to 560 of EphB4 (SEQ ID NO:1). The tyrosinekinase domain corresponds to residues 615 to 874 of EphB4 (SEQ ID NO:1).The SAM (sterile alpha motif) domain corresponds to residues 904 to 971of EphB4 (SEQ ID NO:1). The PDZ domain corresponds to residues 985 to987 of EphB4 (SEQ ID NO:1).

The placements of these domains relative to the EphB4 amino acidsequence is based on information taken from the most recent report fromNCBI Accession number NP_(—)004435. The N-19 Antibody maps to theN-terminal first 19 amino acids of the sequence which is likely to beamino acids residues 16 to 34 of the precursor EphB4 (SEQ ID NO:1). TheC-16 antibody is directed to the tyrosine kinase domain. The H-200antibody is specifically directed to residues 201 to 400 of EphB4 (SEQID NO:1) in the extracellular domain spanning the cysteine rich regionand the fibronectin domain.

FIG. 20 shows a sequence of a Peptide 11 (SEQ ID NO:12) designed toinclude the proposed epitope sequence and a Peptide 10 (SEQ ID NO:11) inwhich the amino acid Aspartate (D) which carries a charge in thiswild-type sequence is substituted with an uncharged amino acid with asimilar side chain structure Asparagine (N). The numbers and thesequence in bold font refer to the position of the amino acids in theprecursor EphB4 protein.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect the present invention provides a method for inhibitingcancerous growth of a cell, the method comprising contacting the cellwith at least one antibody or an antigen-binding portion thereof,wherein the antibody or antigen-binding portion thereof binds to anepitope located within residues 200 to 400 of EphB4 (SEQ ID NO: 1), or asequence at least 85%, preferably at least 90% identical thereto.Preferably, the antibody or antigen-binding portion thereof binds to anepitope located within residues 201 to 245 of EphB4 (SEQ ID NO: 1), or asequence at least 85%, preferably at least 90% identical thereto. Mostpreferably, the antibody or antigen-binding portion thereof binds to anepitope located within residues 220 to 230 of EphB4 (SEQ ID NO: 1), or asequence at least 85%, preferably at least 90% identical thereto.Preferably, the sequence has a substitution of amino acid Asp (D) to Asn(N) at residue 226 of EphB4 (SEQ ID NO: 1).

The antibody or an antigen-binding portion thereof preferablyspecifically binds to a polypeptide having a sequence consisting ofresidues 200 to 400 of EphB4 (SEQ ID NO:1). Preferably, the antibody orantigen-binding portion specifically binds to a polypeptide having asequence consisting of residues 201 to 245 of EphB4 (SEQ ID NO: 1), or asequence at least 85%, preferably at least 90% identical thereto.Preferably, the antibody or antigen-binding portion specifically bindsto a polypeptide having a sequence consisting of residues 220 to 244 ofEphB4 (SEQ ID NO: 1), or a sequence at least 85%, preferably at least90% identical thereto. Most preferably, the antibody or antigen-bindingportion thereof specifically binds to a polypeptide having a sequenceconsisting of residues 220 to 230 of EphB4 (SEQ ID NO: 1), or a sequenceat least 85%, preferably at least 90% identical thereto. Preferably, thesequence has a substitution of amino acid Asp (D) to Asn (N) at residue226 of EphB4 (SEQ ID NO: 1). Most preferably, the antibody or anantigen-binding portion thereof is a polyclonal or monoclonal antibody.The method preferably results in the death of the cell.

The antibody or antigen-binding portion thereof preferably specificallybinds to a polypeptide having a sequence at least 85%, preferably atleast 90% identical to sequence selected from the group consisting ofresidues 200 to 400 of EphB4 (SEQ ID NO:1), residues 201 to 245 of EphB4(SEQ ID NO: 1), residues 220 to 244 of EphB4 (SEQ ID NO: 1) and residues220 to 230 of EphB4 (SEQ ID NO: 1). A polypeptide having a sequence atleast 85%, preferably at least 90% identical to residues 200 to 400 ofEphB4 (SEQ ID NO:1), residues 201 to 245 of EphB4 (SEQ ID NO: 1),residues 220 to 244 of EphB4 (SEQ ID NO: 1) or residues 220 to 230 ofEphB4 (SEQ ID NO: 1), preferably includes polypeptide variants having atleast one substitution, deletion or addition of particular aminoacids(s). Such polypeptide variants are also suitable for the presentmethods, particularly if they retain antigenic properties. For instance,the polypeptide variants can be designed to retain antigenic propertiesand to improve polypeptide production and/or solubility.

For example, antigenic prediction programs suggest that the chargedamino acid Asp (D) may also be important to the epitope function as itis the only charged residue in the sequence. Peptide 11 consisting ofamino acid residues 220 to 244 of EphB4 protein (SEQ ID NO.1) wasdesigned as indicated in FIG. 20. Peptide 10 with a substitution of Asn(N) at residue 226 of EphB4 protein (SEQ ID NO.1) was also designed asindicated in FIG. 20). The amino acid sequence of Peptide 10 and Peptide11 is as follows:

Peptide 10 SEQ ID NO: 11: AGSCVVNAVPAPGPSPSLYCREDGQ Peptide 11 SEQ IDNO: 12: AGSCVVDAVPAPGPSPSLYCREDGQ

The side chains of Asp and Asn are very similar—the hydoxyl group of Aspis an amine in Asn and changes it from being a negatively charged aminoacid to a neutral one.

In a second aspect the present invention also provides a method forinducing cell death of a cancer cell, the method comprising contactingthe cell with at least one antibody or an antigen-binding portionthereof, wherein the antibody or antigen-binding portion thereof bindsto an epitope located within residues 200 to 400 of EphB4 (SEQ ID NO:1), or a sequence at least 85%, preferably at least 90% identicalthereto. Preferably, the antibody or antigen-binding portion thereofbinds to an epitope located within residues 201 to 245 of EphB4 (SEQ IDNO: 1), or a sequence at least 85%, preferably at least 90% identicalthereto. Preferably, the antibody or antigen-binding portion thereofbinds to an epitope located within residues 220 to 244 of EphB4 (SEQ IDNO: 1), or a sequence at least 85%, preferably at least 90% identicalthereto. Most preferably, the antibody or antigen-binding portionthereof binds to an epitope located within residues 220 to 230 of EphB4(SEQ ID NO: 1), or a sequence at least 85%, preferably at least 90%identical thereto. Preferably, the sequence has a substitution of aminoacid Asp (D) to Asn (N) at residue 226 of EphB4 (SEQ ID NO: 1).

In a third aspect the present invention provides a method for treatingor preventing cancer in a subject, the method comprising administeringto the subject an effective amount of at least one antibody or anantigen-binding portion thereof, wherein the antibody or antigen-bindingportion thereof binds to an epitope located within residues 200 to 400of EphB4 (SEQ ID NO: 1), or a sequence at least 85%, preferably at least90% identical thereto. Preferably, the antibody or antigen-bindingportion thereof binds to an epitope located within residues 201 to 245of EphB4 (SEQ ID NO: 1), or a sequence at least 85%, preferably at least90% identical thereto. Preferably, the antibody or antigen-bindingportion thereof binds to an epitope located within residues 201 to 245of EphB4 (SEQ ID NO: 1), or a sequence at least 85%, preferably at least90% identical thereto. Most preferably, the antibody or antigen-bindingportion thereof binds to an epitope located within residues 220 to 230of EphB4 (SEQ ID NO: 1), or a sequence at least 85%, preferably at least90% identical thereto. Preferably, the sequence has a substitution ofamino acid Asp (D) to Asn (N) at residue 226 of EphB4 (SEQ ID NO: 1).

The cancer is preferably selected from the group consisting of breastcancer, prostate cancer, bowel cancer, bladder cancer, colon cancer,ovarian cancer, lung cancer, melanoma, lymphoma and leukemia. The methodpreferably results in the death of a cancer cell in the subject.

In another aspect of the invention there is provided a method ofidentifying an agent which inhibits cancerous growth of a cell, themethod comprising assessing the ability of the agent to bind to an EphB4polypeptide within the region of 200 to 400 of EphB4 (SEQ ID NO:1), or asequence at least 85%, preferably at least 90% identical thereto.

In a preferred embodiment of the invention, the agent binds to anepitope contained within residues 200 to 400 of EphB4 (SEQ ID NO:1), ora sequence at least 85%, preferably at least 90% identical thereto.Preferably, the agent binds to an epitope contained within residues 201to 245 of EphB4 (SEQ ID NO: 1), or a sequence at least 85%, preferablyat least 90% identical thereto. Preferably, the antibody orantigen-binding portion thereof binds to an epitope located withinresidues 201 to 245 of EphB4 (SEQ ID NO: 1), or a sequence at least 85%,preferably at least 90% identical thereto. Most preferably, the agentbinds to an epitope contained within residues 220 to 230 of EphB4 (SEQID NO: 1), or a sequence at least 85%, preferably at least 90% identicalthereto. Preferably, the sequence has a substitution of amino acid Asp(D) to Asn (N) at residue 226 of EphB4 (SEQ ID NO: 1).

The present invention also provides an agent identified by the methoddescribed above.

In the present specification the term “antibody” is used in the broadestsense and specifically covers monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies), andantibody fragments. The term “epitope” refers to an epitope region of apolypeptide that is recognized by an antibody or an antigen bindingportion thereof.

Antibodies refer to immunoglobulin molecules comprised of fourpolypeptide chains, two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain is comprised of aheavy chain variable region (HCVR or VH) and a heavy chain constantregion. The heavy chain constant region is comprised of three domains,CH1, CH2 and CH3. Each light chain is comprised of a light chainvariable region (LCVR or VL) and a light chain constant region. Thelight chain constant region is comprised of one domain, CL. The VH andVL regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR, CDR1,FR2, CDR2, FR3, CDR3, FR4.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein refers to one or more fragments of an antibodythat retains the ability to specifically bind to an antigen. It has beenshown that the antigen-binding function of an antibody can be performedby fragments of a full length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibodyinclude (I) a Fab fragment, a monovalent fragment consisting of the VL,VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody; (v) a dAb fragment (8) which consists of a VH domain, or a VLdomain (9); and (vi) an isolated complementarity determining region(CDR). Furthermore, although the two domains of the Fv fragment, VL andVH, are coded by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv)(10), (11). Such single chainantibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. Other forms of single chainantibodies, such as diabodies or triabodies are also encompassed.Diabodies are bivalent, bispecific antibodies in which VH and VL domainsare expressed on a single polypeptide chain, but using a linker that istoo short to allow for pairing between the two domains on the samechain, thereby forcing the domains to pair with complementary domains ofanother chain and creating two antigen binding sites (12, 13).Preferably, the antibody is EphB4 (H-200) rabbit polyclonal Ig Gantibody, Santa Cruz Biotechnology, Santa Cruz, Calif.

More preferably, the antibody is a monoclonal antibody or fragmentthereof and, particularly, is selected from monoclonal antibodies orfragments thereof which bind to an epitope within residues 200 to 400 ofEphB4 (SEQ ID NO:1), or a sequence at least 85%, preferably at least 90%identical thereto. Preferably, the monoclonal antibodies or fragmentsthereof bind to an epitope within residues 201 to 245 of EphB4 (SEQ IDNO: 1), or a sequence at least 85%, preferably at least 90% identicalthereto. Preferably, the monoclonal antibodies or fragments thereof bindto an epitope within residues 220 to 244 of EphB4 (SEQ ID NO: 1), or asequence at least 85%, preferably at least 90% identical thereto. Mostpreferably, the monoclonal antibodies or fragments thereof bind to anepitope within residues 220 to 230 of EphB4 (SEQ ID NO: 1), or asequence at least 85%, preferably at least 90% identical thereto.Preferably, the sequence has a substitution of amino acid Asp (D) to Asn(N) at residue 226 of EphB4 (SEQ ID NO: 1).

In a further aspect of the invention there is provided a purified EphB4antibody which binds to a polypeptide having a sequence at least 85%identical to residues 201 to 245 of EphB4 (SEQ ID NO: 1), preferably atleast 90% identical to residues 201 to 245 of EphB4 (SEQ ID NO: 1).Preferably, the purified EphB4 antibody binds to a polypeptide having asequence at least 85% identical to residues 220 to 244 of EphB4 (SEQ IDNO: 1), preferably at least 90% identical to residues 220 to 244 ofEphB4 (SEQ ID NO: 1). The purified EphB4 antibody preferably binds to apolypeptide having a sequence at least 85% identical to residues 220 to230 of EphB4 (SEQ ID NO: 1), preferably at least 90% identical toresidues 220 to 230 of EphB4 (SEQ ID NO: 1). More preferably, thepresent invention provides a purified EphB4 antibody which binds to anepitope located in residues 200 to 400 of EphB4 (SEQ ID NO: 1). Thepurified EphB4 antibody according to the present invention preferablybinds to a polypeptide having a substitution of amino acid Asp (D) toAsn (N) at residue 226 of EphB4 (SEQ ID NO: 1). More preferably, thepurified EphB4 antibody is a monoclonal antibody.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method, isolated from phage antibody libraries, ormay be made by recombinant DNA methods. Such techniques include, but arenot restricted to, the hybridoma technique (14), the trioma technique,the human B-cell hybridoma technique (15), and the EBV hybridomatechnique to produce human monoclonal antibodies (16). In addition,humanised monoclonal antibodies can be generated according to methodsdescribed in U.S. Pat. No. 6,090,382 of which the entire description andreferences cited therein are incorporated herein. The document providessuitable host cells for expressing recombinant human antibodies andmethods of synthesising the recombinant human antibodies. Furthermore,suitable human antibodies may be produced using transgenic animals usingfor example techniques described in Oncology 29 (Supp 4) 47-50 (2002).The antibodies of the present invention may also be obtained fromcommercial sources.

Various procedures known in the art may also be used for the productionof polyclonal antibodies which can bind to a polypeptide having asequence comprising residues 200 to 400 of EphB4 (SEQ ID NO:1), or asequence at least 85%, preferably at least 90% identical thereto. Forproduction of the antibodies, various host animals can be immunized byinjection with a EphB4 protein or a EphB4 polypeptide fragment bound toa suitable carrier. Suitable carriers can include, but are not limitedto, BSA (bovine serum albumin), KLH (keyhole limpet hemocyanin), OVA(ovalbumin), THY (Thyroglobulin) and RSA (rabbit serum albumin). Thehost animal is preferably immunized with a EphB4 polypeptide comprisingresidues 200 to 400 of EphB4 (SEQ ID NO:1), or a sequence at least 85%,preferably at least 90% identical thereto. Preferably, the host animalscan be immunized by injection with a EphB4 protein or a polypeptidecomprising residues 201 to 245 of EphB4 (SEQ ID NO: 1), or a sequence atleast 85%, preferably at least 90% identical thereto. Preferably, thehost animals can be immunized by injection with a EphB4 protein or apolypeptide comprising residues 220 to 244 of EphB4 (SEQ ID NO: 1), or asequence at least 85%, preferably at least 90% identical thereto. Mostpreferably, the host animals can be immunized by injection with a EphB4protein or a polypeptide comprising residues 220 to 230 of EphB4 (SEQ IDNO: 1), or a sequence at least 85%, preferably at least 90% identicalthereto. Preferably, the sequence has a substitution of amino acid Asp(D) to Asn (N) at residue 226 of EphB4 (SEQ ID NO: 1).

Suitable host animals include, but are not limited to, rabbits, mice,rats, etc. Various adjuvants can be used to increase the immunologicalresponse, depending on the host species, and include, but are notlimited to, Freud's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol,and potentially useful human adjuvants such as Badilus Calmette-Guerin(BCG) and Corynebacterium parvum. Antibodies and antibody fragments maybe produced in large amounts by standard techniques (eg in either tissueculture or serum free using a fermenter) and purified using affinitycolumns such as protein A (eg for murine Mabs), Protein G (eg for ratMabs) or MEP HYPERCEL (eg for IgM and IgG Mabs).

Suitable antibodies may include antibody fragments that include anantigen-binding portion that can bind to a polypeptide having a sequencecomprising residues 200 to 400 of EphB4 (SEQ ID NO:1), or a sequence atleast 85%, preferably at least 90% identical thereto. Theantigen-binding portion of an antibody preferably includes idiotypes ofresidues 200 to 400 of EphB4 (SEQ ID NO:1), or a sequence at least 85%,preferably at least 90% identical thereto. Preferably, the antibodyfragments include an antigen-binding portion that can bind to apolypeptide having a sequence comprising residues 201 to 245 of EphB4(SEQ ID NO: 1), or a sequence at least 85%, preferably at least 90%identical thereto. Preferably, the antibody fragments include anantigen-binding portion that can bind to a polypeptide having a sequencecomprising residues 220 to 244 of EphB4 (SEQ ID NO: 1), or a sequence atleast 85%, preferably at least 90% identical thereto. Most preferably,the antibody fragments include an antigen-binding portion that can bindto a polypeptide having a sequence comprising residues 220 to 230 ofEphB4 (SEQ ID NO: 1), or a sequence at least 85%, preferably at least90% identical thereto. Preferably, the sequence has a substitution ofamino acid Asp (D) to Asn (N) at residue 226 of EphB4 (SEQ ID NO: 1).

Such antibody fragments can be generated by techniques known in the art.For example, such fragments include, but are not limited to, the F(ab′)₂ fragment which can be produced by pepsin digestion of theantibody molecule; the Fab′ fragments that can be generated by reducingthe disulfide bridges of the F (ab′)2 fragment, the Fab fragments thatcan be generated by treating the antibody molecule with papain and areducing agent, and Fv fragments. In a further technique, recombinantantibodies specific to a polypeptide having a sequence comprisingresidues 200 to 400 of EphB4 (SEQ ID NO:1), preferably, residues 201 to245 of EphB4 (SEQ ID NO:1), preferably, residues 220 to 244 of EphB4(SEQ ID NO:1), more preferably, residues 220 to 230 of EphB4 (SEQ IDNO:1), or a sequence at least 85%, preferably at least 90% identicalthereto, can be engineered and ectopically expressed in a wide varietyof cell types. Preferably, the sequence has a substitution of amino acidAsp (D) to Asn (N) at residue 226 of EphB4 (SEQ ID NO: 1).

The antibodies used in the present methods can include “humanized” formsof non-human (eg., murine) antibodies that are immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′).sub.2 or other antigen-binding subsequences of antibodies) whichcontain minimal amino acid residues derived from a non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human FRresidues. Furthermore, a humanized antibody may comprise residues whichare found neither in the recipient antibody nor in the imported CDR orframework sequences. These modifications can be made to further refineand optimize antibody performance.

The term “EphB4 protein” as used herein is taken to include full lengthEphB4 protein or a polypeptide fragment that comprises residues 200 to400 of EphB4 (SEQ ID NO:1), or a sequence at least 85%, preferably atleast 90% identical thereto. Preferably, the EphB4 protein includes apolypeptide fragment that comprises residues 201 to 245 of EphB4 (SEQ IDNO: 1), or a sequence at least 85%, preferably at least 90% identicalthereto. Preferably, the EphB4 protein includes a polypeptide fragmentthat comprises residues 220 to 244 of EphB4 (SEQ ID NO: 1), or asequence at least 85%, preferably at least 90% identical thereto. Mostpreferably, the EphB4 protein includes a polypeptide fragment thatcomprises residues 220 to 230 of EphB4 (SEQ ID NO: 1), or a sequence atleast 85%, preferably at least 90% identical thereto. Preferably, thesequence has a substitution of amino acid Asp (D) to Asn (N) at residue226 of EphB4 (SEQ ID NO: 1).

A EphB4 variant protein may been modified at the amino acid level andmay include additions or deletions or replacements of amino acids whichdo not affect the functionality of the protein, such as conservativeamino acid substitutions. An EphB4 protein may also include a truncatedEphB4 protein. An EphB4 protein may be natural or recombinant. The EphB4protein may be from any animal species, preferably the EphB4 protein ishuman.

An antibody or an antigen-binding portion thereof that is suitable forthe methods of the present invention, preferably can inhibit cancerousgrowth of a cell by inhibiting the activity of an EphB4 protein. In thespecification the term “cancerous growth” is taken to refer to abnormaland uncontrollable division and growth of a cell. Typically such a cellis identified as a cancer cell that may be able to invade and disruptother tissues and has the potential to spread to other areas of thebody. Cancerous growth of a cell can lead to the formation of a tumorthat may be benign or malignant.

In the specification the term “cell(s)” is taken to include any cells.Preferably, the cells are derived from a mammalian species, such as, butnot limited to, human, mice, bovine, sheep or domestic animals. It ispreferred that the cells are selected from the group including, but notlimited to, prostate cells, breast cells, colon cells, fibroblasts,epidermal cells, placental, liver, kidney, pancreas, heart, neural ormuscle cells, or cancer or tumor cells. The cells may be normal cells,diseased cells, adult cells or embryonic cells. The cells may be singlecells, cultured cells or part of a tissue. The cells may be geneticallymodified recombinant cells, such as a transgenic cell. Preferably, thecells express EphB4. The cells may be part of a whole animal. The cellsmay also be derived from a cell line. Preferably, the cells are from acell line derived from, but not limited to, prostate, breast, colon orovary cell line. The cell line is preferably selected from the groupconsisting of colon SW480, colon SW620, colon LIM1215, breast MCF7,breast T47-D, breast MDA-MB-231, breast MDA-MB-453, bladder J82, bladderT24, bladder RT119 and bladder 5637. More preferably, the cell line isselected from the group consisting of breast cancer cell line MCF-7 andcolon cancer cell line SW480.

The antibody or an antigen-binding portion thereof of the presentinvention preferably can inhibit cancerous growth of one or more ofcancer cells selected from the group consisting of breast cancer cells,prostate cancer cells, bowel cancer cells, bladder cancer cells, coloncancer cells, ovarian cancer cells, lung cancer cells, melanoma cells,lymphoma cells and leukemia cells.

The antibody or an antigen-binding portion thereof preferablyspecifically binds to a polypeptide having a sequence comprisingresidues 200 to 400 of EphB4 (SEQ ID NO:1), preferably residues 201 to245 of EphB4 (SEQ ID NO:1), preferably residues 220 to 244 of EphB4 (SEQID NO:1), more preferably residues 220 to 230 of EphB4 (SEQ ID NO:1), ora sequence at least 85%, preferably at least 90% identical thereto. In apreferred embodiment of the invention, at least one antibody or anantigen-binding portion thereof specifically binds to an epitopecontained within residues 200 to 400 of EphB4 (SEQ ID NO:1), preferablyresidues 201 to 245 of EphB4 (SEQ ID NO:1), preferably residues 220 to244 of EphB4 (SEQ ID NO:1), more preferably residues 220 to 230 of EphB4(SEQ ID NO:1), or a sequence at least 85%, preferably at least 90%identical thereto.

The term “specifically binds” in this specification, is to be understoodto refer to binding characteristics of an antibody or an antigen-bindingportion thereof which binds exclusively to a polypeptide having asequence comprising residues 200 to 400 of EphB4 (SEQ ID NO:1),preferably residues 201 to 245 of EphB4 (SEQ ID NO:1), preferablyresidues 220 to 244 of EphB4 (SEQ ID NO:1), more preferably residues 220to 230 of EphB4 (SEQ ID NO:1), or a sequence at least 85%, preferably atleast 90% identical thereto. The antibody or an antigen-binding portionthereof is preferably a polyclonal or monoclonal antibody thatspecifically binds to a polypeptide having a sequence comprisingresidues 200 to 400 of EphB4 (SEQ ID NO:1), preferably residues 201 to245 of EphB4 (SEQ ID NO:1), preferably residues 220 to 244 of EphB4 (SEQID NO:1), more preferably residues 220 to 230 of EphB4 (SEQ ID NO:1), ora sequence at least 85%, preferably at least 90% identical thereto.Preferably, the antibody is an EphB4 polyclonal antibody (H-200-SantaCruz Biotechnology) specifically directed to the extracellular domainamino residues 201 to 400 of EphB4 (SEQ ID NO:1).

The present invention provides a method for inhibiting cancerous growthof a cell, the method comprising contacting the cell with at least oneantibody or an antigen-binding portion thereof, wherein the antibody orantigen-binding portion thereof binds to an epitope located withinresidues 200 to 400 of EphB4 (SEQ ID NO:1), preferably residues 201 to245 of EphB4 (SEQ ID NO:1), preferably residues 220 to 244 of EphB4 (SEQID NO:1), more preferably residues 220 to 230 of EphB4 (SEQ ID NO:1), ora sequence at least 85%, preferably at least 90% identical thereto.Preferably, the sequence has a substitution of amino acid Asp (D) to Asn(N) at residue 226 of EphB4 (SEQ ID NO: 1).

Preferably, at least one antibody or an antigen-binding portion thereofbinds to a polypeptide having a sequence comprising residues 201 to 400of EphB4 (SEQ ID NO:1), or a sequence at least 85%, preferably at least90% identical thereto. Preferably, the antibody or an antigen-bindingportion thereof binds to a EphB4 protein having a sequence comprisingresidues 201 to 245 of EphB4 (SEQ ID NO:1), or a sequence at least 85%,preferably at least 90% identical thereto. Preferably, the antibody oran antigen-binding portion thereof binds to a EphB4 protein having asequence comprising residues 220 to 244 of EphB4 (SEQ ID NO:1), or asequence at least 85%, preferably at least 90% identical thereto. Morepreferably, the antibody or an antigen-binding portion thereof binds toa EphB4 protein having a sequence comprising residues 220 to 230 ofEphB4 (SEQ ID NO:1), or a sequence at least 85%, preferably at least 90%identical thereto. Preferably, the sequence has a substitution of aminoacid Asp (D) to Asn (N) at residue 226 of EphB4 (SEQ ID NO: 1).

The phrase “inhibiting cancerous growth of a cell” as used herein istaken to mean that cancerous growth of the cell is substantially reducedor prevented. In the present invention a cell is contacted with at leastone antibody or an antigen-binding portion thereof which binds to apolypeptide having a sequence comprising residues 200 to 400 of EphB4(SEQ ID NO:1), preferably residues 201 to 245 of EphB4 (SEQ ID NO:1),preferably residues 220 to 244 of EphB4 (SEQ ID NO:1), more preferablyresidues 220 to 230 of EphB4 (SEQ ID NO:1), or a sequence at least 85%,preferably at least 90% identical thereto, to result in the inhibitionof cancerous growth of the cell as compared to an untreated cell. Themethod preferably results in the death of the cell. Preferably, thesequence has a substitution of amino acid Asp (D) to Asn (N) at residue226 of EphB4 (SEQ ID NO: 1).

The present invention also provides a method for inducing cell death ofa cancer cell, the method comprising contacting the cell with at leastone antibody or an antigen-binding portion thereof which binds to apolypeptide having a sequence comprising residues 200 to 400 of EphB4(SEQ ID NO:1), or a sequence at least 85%, preferably at least 90%identical thereto. In a preferred embodiment of the invention, at leastone antibody or an antigen-binding portion thereof binds to an epitopecontained within residues 201 to 245 of EphB4 (SEQ ID NO:1), or asequence at least 85%, preferably at least 90% identical thereto.Preferably, at least one antibody or an antigen-binding portion thereofbinds to an epitope contained within residues 220 to 244 of EphB4 (SEQID NO:1), or a sequence at least 85%, preferably at least 90% identicalthereto. More preferably, at least one antibody or an antigen-bindingportion thereof binds to an epitope contained within residues 220 to 230of EphB4 (SEQ ID NO:1), or a sequence at least 85%, preferably at least90% identical thereto. Preferably, the sequence has a substitution ofamino acid Asp (D) to Asn (N) at residue 226 of EphB4 (SEQ ID NO: 1).

The phrase “inducing cell death of a cancer cell” is taken to mean thata cancer cell contacted with at least one antibody or an antigen-bindingportion thereof which binds to a polypeptide having a sequencecomprising residues 200 to 400 of EphB4 (SEQ ID NO:1), preferablyresidues 201 to 245 of EphB4 (SEQ ID NO:1), preferably residues 220 to244 of EphB4 (SEQ ID NO:1), more preferably residues 220 to 230 of EphB4(SEQ ID NO:1), or a sequence at least 85%, preferably at least 90%identical thereto, is caused to undergo cell death. Preferably, theantibody is an EphB4 polyclonal antibody (H-200-Santa CruzBiotechnology) specifically directed to the extracellular domain aminoresidues 201 to 400 of EphB4 (SEQ ID NO:1).

Cell death of a cancer cell may be assessed by a number of assays. Forexample, caspase-3 activation is considered to play a key role in theinitiation of cellular events during cell death. Many different kits forthe quantification of caspase-3 activity are available commercially.Mitochondrial membrane depolarization is often associated with the earlystage of cell death. Changes in the membrane potential are presumed tobe due to the opening of the mitochondrial permeability transitionpores, which may play a central role in apoptosis. Depolarization can bedetected by a number of different assays including the use of Rhodamine123, a green-fluorescent cationic dye that accumulates in activemitochondria, which have high membrane potentials allowing quick andeasy detection o cellular disruption. Lactate dehydrogenase (LDH) is astable cytoplasmic enzyme present in all cells. It is rapidly releasedinto the cell culture supernatant when the plasma membrane is damaged.LDH activity can easily be measured in culture supernatant by a singlepoint assay using a spectrophotometric plate reader using commerciallyavailable kits. Elevated LDH in the culture medium is an indication ofcell necrosis (death).

The morphology of a cell can also be examined to assess cell death. Forinstance, apoptosis is programmed cell death which is characterised by aseries of typical morphological events, such as shrinkage of the celland fragmentation into membrane-bound apoptotic bodies (17). These canbe seen using a light microscope. In addition, a cell can be examinedfor the expression of genes related to cell death. In addition, RT-PCRanalysis comparing EphB4 antibody treated and untreated cells from fourdifferent breast cancer cell lines has shown that EphB4 gene expressionis down-regulated in treated cells.

A further aspect of the present invention is a method for treating orpreventing cancer in a subject, the method comprising administering tothe subject an effective amount of at least one antibody or anantigen-binding portion thereof which binds to a polypeptide having asequence comprising residues 200 to 400 of EphB4 (SEQ ID NO:1),preferably residues 201 to 245 of EphB4 (SEQ ID NO:1), preferablyresidues 220 to 244 of EphB4 (SEQ ID NO:1), more preferably residues 220to 230 of EphB4 (SEQ ID NO:1), or a sequence at least 85%, preferably atleast 90% identical thereto. Preferably, the sequence has a substitutionof amino acid Asp (D) to Asn (N) at residue 226 of EphB4 (SEQ ID NO: 1).The method preferably results in the death of a cancer cell in thesubject.

The cancer is preferably selected from the group consisting of breastcancer, prostate cancer, bowel cancer, bladder cancer, colon cancer,ovarian cancer, lung cancer, melanoma, lymphoma and leukemia.

The subject treated by the methods of the invention may be selectedfrom, but is not limited to, the group consisting of humans, sheep,cattle, horses, bovine, pigs, poultry, dogs and cats.

In the method an effective amount of at least one antibody or anantigen-binding portion thereof which binds to a polypeptide having asequence comprising residues 200 to 400 of EphB4 (SEQ ID NO:1),preferably residues 201 to 245 of EphB4 (SEQ ID NO:1), preferablyresidues 220 to 244 of EphB4 (SEQ ID NO:1), more preferably residues 220to 230 of EphB4 (SEQ ID NO:1), or a sequence at least 85%, preferably atleast 90% identical thereto, is administered to a subject. In apreferred embodiment of the invention, at least one antibody or anantigen-binding portion thereof binds to an epitope contained withinresidues 200 to 400 of EphB4 (SEQ ID NO:1), preferably residues 201 to245 of EphB4 (SEQ ID NO:1), preferably residues 220 to 244 of EphB4 (SEQID NO:1), more preferably residues 220 to 230 of EphB4 (SEQ ID NO:1), ora sequence at least 85%, preferably at least 90% identical thereto. Theantibody or an antigen-binding portion thereof preferably specificallybinds to a polypeptide having a sequence comprising residues 200 to 400of EphB4 (SEQ ID NO:1), preferably residues 201 to 245 of EphB4 (SEQ IDNO:1), preferably residues 220 to 244 of EphB4 (SEQ ID NO:1), morepreferably residues 220 to 230 of EphB4 (SEQ ID NO:1), or a sequence atleast 85%, preferably at least 90% identical thereto. Most preferably,the antibody or an antigen-binding portion thereof is a polyclonal ormonoclonal antibody.

The term “effective amount” means a dosage sufficient to providetreatment or prevention for the cancer being treated or prevented. Thiswill vary depending on the subject and the type of cancer beingeffected. The effective amounts of at least one antibody or anantigen-binding portion thereof used in the methods of the presentinvention may vary depending upon the manner of administration, thecondition of the animal to be treated, and ultimately will be decided bythe attending scientist, physician or veterinarian. The amount ofantibody or an antigen-binding portion thereof used to treat or preventa subject will also vary depending upon the nature and identity of theparticular antibody or an antigen-binding portion thereof.

An antibody or an antigen-binding portion thereof is preferablyadministered to a subject by any suitable means known to those skilledin the art. Preferably, the antibody or an antigen-binding portionthereof can be contacted with a cell in numerous fashions, including,for example, intravenously.

Preferably, the antibody or an antigen-binding portion thereof of thepresent invention is combined with a suitablepharmaceutically-acceptable carrier or diluent to form a pharmaceuticalcomposition which may be suitable for administration to a human oranimal subject. Suitable carriers or diluents include isotonic salinesolutions, for example, phosphate-buffered saline. The pharmaceuticalcomposition including at least one antibody or an antigen-bindingportion thereof may be formulated for parenteral, intramuscular,intravenous, subcutaneous, intraocular, oral or transdermaladministration. The antibody may be administered at a suitable dosedependent on the body weight of the subject. It is to be understood,however, that the routes of administration and dosages mentioned areintended to serve only as a guide since a person skilled in the artwould be able to readily determine the optimum route of administrationand dosage for any particular subject and cancer.

The antibody or an antigen-binding portion thereof used in the methodsof the present invention may be combined with suitable excipients, suchas emulsifiers, surfactants, stabilisers, dyes, penetration enhancers,anti-oxidants, water, salt solutions, alcohols, polyethylene glycols,gelatine, lactose, magnesium sterate and silicic acid. The antibody oran antigen-binding portion thereof is preferably formulated as a sterileaqueous solution. The antibody or an antigen-binding portion thereof canbe combined with adjunct components that are compatible with theactivity of the antibody. An antibody or an antigen-binding portionthereof used in the methods of the present invention may be preferablyused to complement existing treatments for cancer. For example, themethod of the present invention may also be used in combination withtraditional cancer treatments such as radiotherapy, chemotherapy (egusing anthracyclines, 5FU, topoisomerase inhibitors, Cisplatin andCarboplatin), or hormone therapy or therapies utilising hormonemodifiers (eg Catamoxifen).

In another aspect of the invention there is provided a method ofidentifying an agent which inhibits cancerous growth of a cell, themethod comprising assessing the ability of the agent to bind to apolypeptide having a sequence comprising residues 200 to 400 of EphB4(SEQ ID NO:1), preferably residues 201 to 245 of EphB4 (SEQ ID NO:1),preferably residues 220 to 244 of EphB4 (SEQ ID NO:1), more preferablyresidues 220 to 230 of EphB4 (SEQ ID NO:1), or a sequence at least 85%,preferably at least 90% identical thereto. In a preferred embodiment ofthe invention, the agent binds to an epitope contained within residues200 to 400 of EphB4 (SEQ ID NO:1), preferably residues 201 to 245 ofEphB4 (SEQ ID NO:1), preferably residues 220 to 244 of EphB4 (SEQ IDNO:1), more preferably residues 220 to 230 of EphB4 (SEQ ID NO:1), or asequence at least 85%, preferably at least 90% identical thereto.Preferably, the agent binds to a EphB4 protein having a sequencecomprising residues 200 to 400 of EphB4 (SEQ ID NO:1), preferablyresidues 201 to 245 of EphB4 (SEQ ID NO:1), preferably residues 220 to244 of EphB4 (SEQ ID NO:1), more preferably residues 220 to 230 of EphB4(SEQ ID NO:1), or a sequence at least 85%, preferably at least 90%identical thereto.

In the present specification the term “agent” is taken to include anymolecule, compound or protein that can bind (interact with) residues 200to 400 of EphB4 (SEQ ID NO:1), preferably residues 201 to 245 of EphB4(SEQ ID NO:1), preferably residues 220 to 244 of EphB4 (SEQ ID NO:1),more preferably residues 220 to 230 of EphB4 (SEQ ID NO:1), or asequence at least 85%, preferably at least 90% identical thereto.Suitable agents can preferably include an antibody or an antigen-bindingportion thereof that binds to a polypeptide having a sequence comprisingresidues 200 to 400 of EphB4 (SEQ ID NO:1), preferably residues 201 to245 of EphB4 (SEQ ID NO:1), preferably residues 220 to 244 of EphB4 (SEQID NO:1), more preferably residues 220 to 230 of EphB4 (SEQ ID NO:1), ora sequence at least 85%, preferably at least 90% identical thereto. Mostpreferably, the agent is an antibody or an antigen-binding portionthereof that is a polyclonal or monoclonal antibody. The methodpreferably comprises assessing the ability of the agent to induce celldeath of a cancer cell. The agent is preferably a EphB4 ligand, such asan antibody or an antigen-binding portion thereof, that is preferablyspecific for EphB4 protein and may be developed or obtained commerciallyfor testing in in vitro or in vivo systems for its ability to inhibitcancerous growth of a cell.

For instance, antibodies or antigen-binding portions thereof directed tospecific epitopes of residues 200 to 400 of EphB4 (SEQ ID NO:1),preferably residues 201 to 245 of EphB4 (SEQ ID NO:1), preferablyresidues 220 to 244 of EphB4 (SEQ ID NO:1), more preferably residues 220to 230 of EphB4 (SEQ ID NO:1), or a sequence at least 85%, preferably atleast 90% identical thereto, can be tested for their ability to inhibitcancerous growth of a cell and preferably induce cell death. Doseresponse curves to assess the IC50 of the antibodies can be conducted totest efficacy of each antibody tested. In addition,antibody/receptor-ligand binding studies can be performed to assess theability of the antibody to prevent ligand binding. Tyrosinephosphorylation of the EphB4 receptor following antibody binding can beassessed by immunoprecipitation of the receptor with the respectiveantibody, followed by Western analysis with an anti-phosphotyrosineantibody to confirm that the EphB4 receptor is inactivated. The antibodywith the best neutralising activity in terms of inhibiting tyrosinephosphorylation and cell growth in vitro and preventing ligand bindingto the EphB4 receptor at the lowest 50% inhibitory concentration (IC₅₀)can be selected for additional in vivo tests.

For instance, an in vivo model of metastasis and tumour growth usingimmune-deficient NOD-SCID (non-obese diabetic, combinedimmunodeficiency) mice can be used to test the ability of putativeagents that can bind to a polypeptide having a sequence comprisingresidues 200 to 400 of EphB4 (SEQ ID NO:1), preferably residues 201 to245 of EphB4 (SEQ ID NO:1), preferably residues 220 to 244 of EphB4 (SEQID NO:1), more preferably residues 220 to 230 of EphB4 (SEQ ID NO:1), ora sequence at least 85%, preferably at least 90% identical thereto, fortheir efficacy as an anti-cancer agent. Preferably, the sequence has asubstitution of amino acid Asp (D) to Asn (N) at residue 226 of EphB4(SEQ ID NO: 1). Moreover, a diverse array of tumor cell lines that areavailable, most of which can be grown as xenografts, and these includethe human breast cancer cell line MCF-7 and colon cancer cell line HT29,can be used for in vitro testing. Xenograft tumours can be grown in themouse model either after subcutaneous injection, where they will grow asa mass, or after injection into the tail vein allowing mimicry of thehematogenous spread of metastasis that results in secondary deposits inother organs. Once suitable engraftment periods and inoculation dosesfor each cell line have been established, the model can be used to testvarious agents that bind to a polypeptide having a sequence comprisingresidues 200 to 400 of EphB4 (SEQ ID NO:1), preferably residues 201 to245 of EphB4 (SEQ ID NO:1), preferably residues 220 to 244 of EphB4 (SEQID NO:1), more preferably residues 220 to 230 of EphB4 (SEQ ID NO:1), ora sequence at least 85%, preferably at least 90% identical thereto.Cells may also be treated with sub-lethal doses of a chosen EphB4antibody equating to the IC₅₀ and IC₇₅ to assess the engraftment oftreated cells compared with non-treated cells. This will assess theeffects of reduced functional expression of EphB4 on establishment andmetastasis of tumour cell lines.

The in vivo models can also be used for pre-clinical assessment ofpotential new therapies for treatment of EphB4 positive tumours celllines. The use of subcutaneous injection will allow the examination oftumours that have been allowed to establish for different periods oftime. This can be used to determine the ability of an agent, such as anantibody or an antigen-binding portion thereof, to ablate newly andwell-established tumours compared to vehicle control. The use of tailvein injections can be used to determine whether treatment with anantibody or an antigen-binding portion thereof will reduce number ofmetastases formed as a result of hematogenous spread. The agentsidentified by the methods of the present invention may be used fortreatment or prevention of cancer. The present invention also providesan agent identified by the method described above.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

The invention will hereinafter be described by way of the followingnon-limiting Figures and Examples.

EXAMPLE 1 Immunohistochemical Localisation of EphB4

An EphB4 polyclonal antibody (H-200-Santa Cruz Biotechnology)specifically directed to the extracellular domain amino residues 201 to400 of EphB4 (SEQ ID NO:1) was used to analyse the localisation of theEphB4 protein in tumour and normal tissue. Colon and breast tissueshowed marked increase in the levels of this protein in the tumourepithelial cells when compared with the matched normal tissue (as shownin FIG. 1). The demonstration of high expression of EphB4 on the tumourepithelial cells in two of the most commonly occurring cancers suggeststhat EphB4 is critical to the progression of these tumours.

EXAMPLE 2 RT-PCR Expression of EphB4

Reverse transcriptase-polymerase chain reaction (RT-PCR) was used tocompare expression of EphB4(1187 bp) and internal 18S rRNA (489 bp) infive tumour (T)/normal (N) pairs (results shown in FIG. 2). Analysis of63 colon cancers from 60 patients indicated that EphB4 is over-expressedin the tumour tissue of 80% of patients, implying broad application as atherapeutic target (FIG. 2). The differential expression between tumourcells and normal tissue suggests anti-EphB4 tumour therapy may have apreferential effect on colon (and other) tumours.

A comparison of the expression profile of EphB4 with that of otherreceptor protein tyrosine kinases already being targeted in clinicaltrials (HER2, EGFR and VEGFR) suggests that EphB4 is expressed to alesser degree in normal tissues. Information from EST databases suggeststhat low level expression of EphB4 may be present in kidney, ovary andplacenta, and very low level expression in heart, lung, peripheralnerves and vascular tissue. Accordingly therapies that target EphB4 maybe expected to produce less side effects than those that target otherreceptor tyrosine kinases.

EXAMPLE 3 EphB4-Specific Antibody Studies

A direct tumoricidal effect of an EphB4 polyclonal antibody (H-200-SantaCruz Biotechnology) specifically directed to the extracellular domainamino residues 201 to 400 of EphB4 (SEQ ID NO:1) in vitro wasdemonstrated. Incubation of a fully confluent monolayer of colon cancercell lines SW480 and SW620 with a 1/500 dilution of EphB4 antibody H-200(Santa Cruz Biotechnology—Lot numbers B141 and F182) caused the cells tolift of the bottom of the culture vessel and die (FIG. 3). This was acommon response seen when treating colon cancer cell lines includingSW480, SW620, LIM1215, breast cancer cell lines MCF-7, T47-D,MDA-MB-453, MDA-MB-231 and Hs578t, bladder cancer cell lines J82, RT119,5637 and T24.

Incubation of the breast cancer cell line MCF-7 and the colon cancercell line SW480 with three different concentrations of antibody (2μg/ml, 1 μg/μml and 0.2 μg/ml) resulted in cell death in a dosedependent manner (see FIG. 4 and FIG. 5). This effect was not seenfollowing exposure of the endothelial cell line HUVEC-C to the EphB4antibody. Analysis of the caspase-3 activity suggested that cell deathwas not via apoptosis (FIG. 6). These results suggest that bycross-linking or binding to membrane receptors, antibodies may mimic ormodulate receptor activity and that these antibodies-generatedtransmembrane signals might cause apoptosis or growth inhibition (18).Possible alternative mechanisms for the induction of cell death includeras-mediated non-apoptotic cell death or restoration of gap junctionintercellular communication (GJIC), a direct cell-cell communicationpathway that is known to be prevented by Eph receptor signaling (19).

Polyclonal antibodies specific for EphB4 have been developed and areavailable commercially, for testing of these antibodies in vitro and invivo systems. FIG. 7 shows results of the percentage viability of breastcancer cells after treatment with five EphB4 antibodies as detailedbelow:

-   -   (1) a EphB4 polyclonal antibody (Swiss) directed to amino acid        residues 825 to 991 of the carboxy terminus of mouse EphB4;    -   (2) a polyclonal N-terminal EphB4 antibody (N-19 Santa Cruz        Biotechnology) directed to the N-terminal first 19 amino acids        of the EphB4 amino acid sequence which is likely to be amino        acids residues 16 to 34 of the precursor EphB4 (SEQ ID NO:1);    -   (3) a polyclonal EphB4 C-terminal antibody (C-16 Santa Cruz        Biotechnology) directed to the carboxy-terminal corresponding to        tyrosine kinase domain consisting of amino acid residues 615 to        874 of EphB4 (SEQ ID NO:1);    -   (4) a EphB4 polyclonal antibody (H-200-Santa Cruz Biotechnology)        specifically directed to the extracellular domain amino residues        201 to 400 of EphB4 (SEQ ID NO:1);    -   (5) a EphB4 polyclonal antibody (H-200 (old)-Santa Cruz        Biotechnology-Lot number B141 batch) specifically directed to        the extracellular domain amino residues 201 to 400 of EphB4 (SEQ        ID NO:1).

Cell death effect was seen in treatment with the H-200 antibodyspecifically directed to the extracellular domain amino residues 201 to400 of EphB4 (SEQ ID NO:1). A comparison of cells grown in medium withor without active complement proteins shows that complement plays norole in the Ab-mediated cell death (see FIG. 7).

The mechanism of cell death can be further by analysing changes to geneexpression induced in cancer cells in vitro after incubation withsub-lethal doses of the H-200 EphB4 antibody using microarraytechniques.

EXAMPLE 4 Expression of EphB4 in Human Tissue

Information from EST databases and Northern analysis of normal humantissues (20) suggests that low level expression of EphB4 may be presentin kidney, ovary and placenta, and very low level expression in heart,lung, peripheral nerves and vascular tissue, and no expression in brain.In order to determine whether gene expression correlates with the levelof protein actually translated a western analysis using these tissueswas performed with an EphB4 polyclonal antibody (H-200-Santa CruzBiotechnology) specifically directed to the extracellular domain aminoresidues 201 to 400 of EphB4 (SEQ ID NO:1). By comparison with a singlecolon tumour sample it was shown that the level of gene expression doesnot correspond to the amount of EphB4 protein produced in these tissues(FIG. 8). The differential expression between tumour cells and normaltissue suggests that anti-EphB4 tumour therapy may have a preferentialeffect on colon tumours.

Several hybridising bands were obtained with Western blot analysis (datanot shown). However, the identity of these protein bands that arespecific to the tumour sample(s), and common to all tumour samplestested, remains to be determined but may correspond to splice variation.Two EphB4 splice variants (named EphB4v1 and EphB4v2) were identified.Determination of the EphB4 gene structure has shown that EphB4v1 resultsfrom the absence of the 53 amino acids encoded by the entire exon 16(FIG. 9). If EphB4v1 is translated the resultant protein would lack aportion of both the kinase and SAM domains, two protein domains that areknown to have roles in transmitting signals to intracellular targets.EphB4v2 is caused by the in-frame deletion of the entire exon 6 whichresults in the absence of 111 amino acids. EphB4v2would encode a proteinthat lacks the first fibronectin type III repeat. The role offibronectin type III repeats is not known and the effect of removing oneof these repeats is unknown at this time.

EXAMPLE 5 RT-PCR Anlaysis of EphB4

Because of EphB4 possible role in angiogenesis, the expression of EphB4in endothelial cells using RT-PCR was performed. Low level of expressionof EphB4 was observed. However, when these cells were gown in thepresence of the EphB4 polyclonal antibody (H-200-Santa CruzBiotechnology) specifically directed to the extracellular domain aminoresidues 201 to 400 of EphB4 (SEQ ID NO:1) there was no apparent changein cell growth or morphology, even at the highest concentration ofantibody. The effect of the EphB4 antibody on the growth and morphologyof NIH3T3 cells was also tested. There was no morphological or growthresponse (data not shown). The nontumorigenic breast cell line MCF10A,established from mammary tissue from a 36-year old Caucasian patientwith fibrocystic breast disease (21) was also tested and was observed toexpress EphB4 at low levels but did not respond to the anti-EphB4antibody (data not shown). These results, when considered together withresults gained from the western analysis of normal human tissues(Example 5), suggest that tissues that show a high level of EphB4expression will be negatively affected by an EphB4 antibody.

The results indicated that after treatment with the EphB4 polyclonalantibody (H-200-Santa Cruz Biotechnology) specifically directed to theextracellular domain amino residues 201 to 400 of EphB4 (SEQ ID NO:1)the EphB4 gene is down-regulated (FIG. 10). This agrees with the findingthat most antibodies increase RTK down-regulation and internalisation ofthe receptor after blocking the ligand-receptor interaction andinhibiting ligand-induced RTK signaling. This is since certainantibodies can influence tumour growth by altering the intracellularsignaling pattern inside the targeted tumour cell. The analysis ofglobal changes in gene expression in Ab-treated cells compared tountreated cells may identify genes involved in EphB4-related signaling.

EXAMPLE 6 EphB4 Antibody-Epitope Mapping

A EphB4 commercially available H-200 polyclonal Ab from Santa Cruz israised against a recombinant protein corresponding to amino acids201-400 of SEQ:ID:NO: 1 of EphB4 human receptor. The sequence includesthe cysteine-rich domain and the first fibronectin type III repeat andaccordingly it was expected that several different antigenic regionswould be recognised. Six blocking peptides of 25 amino acids (overlap by5 amino acids, offset by 20 amino acids) were designed against specificamino acid residues of EphB4 protein (SEQ ID NO.1) as indicated in FIG.11. The blocking peptides have the following amino acid sequence:

Peptide 1   SEQ ID NO: 2: TVNLTRFPETVPRELVVPVAGSCVV Peptide 2   SEQ IDNO: 3: GSCVVDAVPAPGPSPSLYCREDGQW Peptide 3   SEQ ID NO: 4:EDGQWAEQPVTGCSCAPGFEAAEGN Peptide 4   SEQ ID NO: 5:AAEGNTKCRACAQGTFKPLSGEGSC Peptide 5   SEQ ID NO: 6:GEGSCQPCPANSHSNTIGSAVCQCR Peptide 6   SEQ ID NO: 7:VCQCRVGYFRARTDPRGAPCTTPPS

The peptides were tested for their ability to prevent cell death afterpre-incubation with the EphB4 polyclonal antibody (H-200-Santa CruzBiotechnology) specifically directed to the extracellular domain aminoresidues 201 to 400 of EphB4 (SEQ ID NO:1). A cocktail of all thepeptides was initially tested and successfully prevented cell death asdetermined using trypan blue exclusion (FIG. 12) and caspase-3 activityassays (FIG. 13). The peptides were then tested separately and partialrescue was observed for Peptide 1 (SEQ ID NO:2) and Peptide 2 (SEQ IDNO:3) (see FIG. 14). Peptide 1 and Peptide 2 overlap by five amino acidsresidues (GSCVV designated SEQ ID NO:13) indicating that this amino acidsequence corresponding to residues 221 to 225 of EphB4 (SEQ ID NO:1) ispossibly the core of the reactive epitope.

Because the initial experiment with the cocktail of peptides effectivelycontained twice the molar amount of the GSCVV (SEQ ID NO:13) sequence(ie residues 221 to 225 of EphB4 (SEQ ID NO:1)) a further blockingexperiment in which twice the amount of each of Peptide 1 (SEQ ID NO:2)and Peptide 2 (SEQ ID NO:3) was compared with the initial amount of thepeptides together. All treatments were successful in preventing tumourcell death caused by the EphB4 polyclonal antibody (H-200 from SantaCruz) (see FIG. 15).

Three new peptides (Peptides 7 to 9) of different lengths that span theGSCVV (SEQ ID NO:13) core epitope sequence were made commercially basedon specific amino acid residues of EphB4 protein (SEQ ID NO.1) asindicated in FIG. 16. The blocking peptides have the following aminoacid sequence:

Peptide 7 SEQ ID NO: 8 AGSCVVDA Peptide 8 SEQ ID NO: 9 VAGSCVVDAVPeptide 9 SEQ ID NO: 10 LVVPVAGSCVVDAVPA.

However, due to the high number of hydrophobic amino acids in peptides 8and 9, these peptides were not soluble in any solution that could beapplied to cells, thus preventing further testing. However, Peptide 7(SEQ ID NO. 8) with an amino acid sequence corresponding to residues 220to 227 of EphB4 (SEQ ID NO:1) was used in blocking experiments and wasable to rescue cells from the cell death effect caused by the additionof the EphB4-antibody (H-200 from Santa Cruz) alone (see FIG. 17).

All publications discussed above are incorporated herein in theirentirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed in Australia or anyother country before the priority date of each claim of thisapplication.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

REFERENCES

-   1. Australian Institute of Health and Welfare (AIHW) and    Australasian Association of Cancer Registries (AACR) 2000. Cancer in    Australia 1997. AIHW cat no. CAN10. Canberra: AIHW (Cancer Series    no. 15).-   2. Wang et al., 1998, Cell 93:741-753.-   3. Dottori et al., 1998, PNAS 1998 95:13248-13253.-   4. Easty et al., 1999, Int. J. Cancer 84:494-501.-   5. Tickle and Altabef, 1999, Curr. Opin. Genet. Dev. 9:455460.-   6. Oates et al., 1999, Mech. Dev. 83:77-94.-   7. O'Leary and Wilkinson, 1999, Curr. Opin. Neurobiol. 9:65-73.-   8. Ward et al, 1989, Nature 341:544-546.-   9. Van den Beuken T et al, 2001, J. Mol. Biol, 310, 591.-   10. Bird et al, 1988, Science 242:423-426.-   11. Huston et al., 1988 Proc. Natl. Acad. Sci. USA 85:5879-5883.-   12. Holliger, P., et al 1993 Proc. Natl. Acad. Sci. USA    90:6444-6448.-   13. Poljak, R. J., et al. 1994 Structure 2:1121-1123.-   14. Kohler and Milstein (1975, Nature 256: 495-497.-   15. Kozbor et al,. 1983, Immunology Today 4: 72.-   16. Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy,    Alan R. Liss, Inc., pp. 77-96.-   17. Saraste and Pulkki, 2000, Cardiovascular Res. 45:528-53.-   18. Tickle and Altabef (1999) Epithelial cell movements and    interactions in limb, neural crest and vasculature. Curr. Opin.    Genet. Dev. 9: 455-460-   19. Mellitzer et al., 1999, Nature 400:77-81.-   20. Bennett et al (1994) Cloning and characterization of HTK, a    novel transmembrane tyrosine kinase of the EPH subfamily. J Biol.    Chem. 269:14211-14218.-   21. Keydar et al (1979) Establishment and characterization of a cell    line of human breast carcinoma origin. Eur. J. Cancer 15:659-670.

1. A method for inhibiting cancerous growth of a mammalian breast cancercell that expresses EphB4, or a mammalian colon cancer cell thatexpresses EphB4, the method comprising contacting said cell with atleast one antibody or an antigen-binding portion thereof, wherein saidantibody or antigen-binding portion thereof binds an EphB4 epitopelocated within residues 200 to 400 of EphB4 (SEQ ID NO:1), binding saidantibody or said antigen-binding portion thereof to said cell's EphB4,and inhibiting said cancerous growth as a result of said binding.
 2. Themethod according to claim 1, wherein the antibody or antigen-bindingportion thereof binds to an epitope located within residues 201 to 245of EphB4 (SEQ ID NO:1).
 3. The method according to claim 2, wherein theantibody or antigen-binding portion thereof binds to an epitope locatedwithin residues 220 to 244 of EphB4 (SEQ ID NO:1).
 4. The methodaccording to claim 3, wherein the antibody or antigen-binding portionthereof binds to an epitope located within residues 220 to 230 of EphB4(SEQ ID NO:1).
 5. The method of claim 1, wherein said cell is a humancell.
 6. A method for inducing cell death of a mammalian breast cancercell that expresses EphB4, or a mammalian colon cancer cell thatexpresses EphB4, the method comprising contacting said cell with atleast one antibody or an antigen-binding portion thereof, wherein theantibody or antigen-binding portion thereof binds an EphB4 epitopelocated within residues 200 to 400 of EphB4 (SEQ ID NO:1), binding saidantibody or said antigen-binding portion thereof to said cell's EphB4,and inducing said cell death as a result of said binding.
 7. The methodaccording to claim 6, wherein the antibody or antigen-binding portionthereof binds to an epitope located within residues 201 to 245 of EphB4(SEQ ID NO:1).
 8. The method according to claim 7, wherein the antibodyor antigen-binding portion thereof binds to an epitope located withinresidues 220 to 244 of EphB4 (SEQ ID NO:1).
 9. The method according toclaim 8, wherein the antibody or antigen-binding portion thereof bindsto an epitope located within residues 220 to 230 of EphB4 (SEQ ID NO:1).10. The method of claim 6, wherein said cell is a human cell.
 11. Amethod for treating breast cancer or colon cancer in a mammaliansubject, wherein said subject is in need of the inhibition of growth, orinducing the cell death of, of breast cancer cells, or colon cancercells, that express EphB4, the method comprising administering to thesubject an effective amount of at least one antibody or anantigen-binding portion thereof, wherein the antibody or antigen-bindingportion thereof binds to an epitope located within residues 200 to 400of EphB4 (SEQ ID NO:1), contacting said antibody or said antigen-bindingportion thereof with said cell as a result of said administering,binding said antibody or said antigen binding portion thereof to saidcell's EphB4 thereby inhibiting said growth of, or inducing the celldeath of, said cancer cells, and treating or preventing said cancer insaid subject as a result of said binding.
 12. The method according toclaim 11, wherein the antibody or antigen-binding portion thereof bindsto an epitope located within residues 201 to 245 of EphB4 (SEQ ID NO:1).13. The method according to claim 12, wherein the antibody orantigen-binding portion thereof binds to an epitope located withinresidues 220 to 244 of EphB4 (SEQ ID NO:1).
 14. The method according toclaim 13, wherein the antibody or antigen-binding portion thereof bindsto an epitope located within residues 220 to 230 of EphB4 (SEQ ID NO:1).15. The method of claim 11, wherein said subject is human.
 16. Themethod of any one of claims 1, 6 and 11, wherein the amino acid sequenceof said cell's EphB4 comprises that of amino acids 200-400 of SEQ IDNO:1.
 17. The method of any one of claims 1, 6 and 11, wherein saidantibody is a monoclonal antibody.